The present invention generally relates to nucleotide sequences that encode proteins which are essential for bacterial growth. More particularly, the present invention is directed to a GTPase obg gene encoding a GTP-binding protein in Staphylococcal bacterial strains. Specifically, the present invention is directed to a Staphylococcus aureus (xe2x80x9cS. aureusxe2x80x9d) obg gene that is capable of expression in a host cell to produce enzymatically functional S. aureus GTP-binding protein. Additionally, the present invention pertains to recombinant expression vectors incorporating the GTPase obg gene of the present invention. The present invention is further directed to Staphylococcal GTP-binding protein, methods for producing GTP-binding protein, and methods for using GTP-binding protein as a novel thereapeutic target in affinity-based pharmacological screening procedures for the discovery of antibiotics active against S. aureus and other Staphylococcal bacteria.
Numerous pathogenic organisms are responsible for infectious disease and health-related problems in humans and other animals throughout the United States and the world. As treatments are developed for combating a particular organism, such as treatments incorporating newly developed antibiotics and chemical compounds effective at eliminating existing strains of a particular organism, newer strains of such organisms emerge which are resistant to the existing treatments. Accordingly, there remains a continual need for the development of new ways for pharmaceutically combating pathogenic organisms.
One particular organism of concern is the bacterium S. aureus, which is an opportunistic human pathogen both in the community and in hospitals, and is the primary cause of nosocomial bacterial infections in the United States. S. aureus has a highly invasive nature and is associated with a number of life threatening systemic illnesses, such as bacteremia/sepsis, toxic shock syndrome and toxic epidermal necrolysis, as well as common bacterial infections of the skin. Once the organism enters the bloodstream, patients are at risk of developing serious diseases such as endocarditis, osteomyelitis, and septic shock.
Despite the development and use of newer antimicrobial agents to combat S. aureus infections, the morbidity and mortality from serious S. aureus infections remain high. One reason is that S. aureus is adept in developing resistance to multiple antibiotics. The recent emergence of methicillin-resistant and vancomycin-resistant strains of S. aureus in Japan, and subsequently in the United States, has further highlighted the importance of finding alternative approaches to the prevention and treatment of Staphylococcal infections, and has focused renewed attention on the need for development of new classes of antibiotics to combat such bacterial strains. Despite the imminent crisis in S. aureus antibiotic resistance, the identification of novel targets for the development of novel antimicrobial agents remains elusive.
One promising way of pharmaceutically combating bacterial strains, including S. aureus and other Staphylococcal strains, is to interfere with genetic processes relating to growth and/or viability of the bacteria. Methods for combating organisms by interfering with genetic processes essential to survival and growth of the organism are becoming of increasing interest. In particular, researchers are directing their attention to chemical compounds that interfere with such processes.
A potential target for use with screening processes to identify chemical compounds that are useful in combating pathogenic organisms is a GTPase superfamily of GTP(guanosinetriphosphate)-binding proteins that includes G-proteins, elongation factors in E. coli, mammalian Ras, and procaryotic proteins such as Era, FtsZ, and Fth, etc. These GTPase regulatory molecules are classified as belonging to the GTPase superfamily due to a common ability to bind guanine nucleotides and hydrolyse GTP. March, xe2x80x9cMembrane-Associated GTPases in Bacteriaxe2x80x9d, Molec. Microbiol., Vol. 6, pp. 1253-57, 1992.
GTP-binding proteins are important signaling molecules in bacteria as well as in eukaryotic cells. GTP-binding proteins have been recognized for many years as components of signal transduction pathways in eukaryotes. Only recently, however, has it been discovered that prokaryotes contain GTP-binding proteins that are essential for growth and/or viability of the organism. The involvement of these bacterial proteins in signal transduction in prokaryotes, however, is still not entirely clear.
One member of this superfamily of GTP-binding proteins which is of particular interest is the protein expressed by the obg gene (short for spoOB-associated GTP-binding protein). The obg gene specifically encodes a GTP-binding protein which is essential for bacterial growth and which is structurally conserved across an extraordinarily wide range of bacterial species. Obg was initially identified as a gene dowstream of the stage 0 sporulation gene spoOB in Bacillus subtilis in 1989. Trach et al., xe2x80x9cThe Bacillus subtilis spoOB Stage 0 Sporulation Operon Encodes An Essential GTP-Binding Proteinxe2x80x9d J. Bacteriol., Vol. 171, pp. 1362-71, 1989. Transcription analysis of this operon revealed that spoOB and obg are cotranscribed.
Various observations have been made about the Obg protein in certain organisms. Obg in Bacillus subtilis has been shown to bind GTP by the cross-linking method. Trach et al., supra. Bacillus subtilis Obg has also been characterized by its enzymatic activity with respect to GTP hydrolysis. Welsh et al., xe2x80x9cBiochemical Characterization of the Essential GTP-Binding Protein Obg of Bacillus subtilisxe2x80x9d, J. Bacteriol., Vol. 176, pp. 7161-68, 1994. It has also been demonstrated that Obg plays a crucial role in sporulation induction in Bacillus subtilis and Streptomyces griseus. Kok et al., xe2x80x9cEffects on Bacillus subtilis of a Conditional Lethal Mutation in the Essential GTP-Binding Protein Obgxe2x80x9d, J. Bacteriol., Vol. 176, pp. 7155-60, 1994; Okamoto et al., xe2x80x9cMolecular Cloning and Characterization of the obg Gene of Streptomyces griseus in Relation to the Onset of Morphological Differentiationxe2x80x9d, J. Bacteriol., Vol. 179, pp. 170-79, 1997.
Very little is known, however, about the physiological function of Obg. Obg homologs have recently been discovered in a diverse range of organisms ranging from bacteria to archaea to humans, and the evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins has a fundamental, but unknown, cellular function. It has been proposed that, by monitoring the intracellular GTP pool size, Obg is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation. Okamoto et al., supra.
Obg is a unique GTPase in that it possesses an extended N-terminal glycine-rich domain not found in eukaryotic or archaeal homologs. An isolated Bacillus subtilus temperature-sensitive obg mutant was found to carry two closely linked missense mutations in the N-terminal domain, suggesting that this portion of obg is essential for cellular function. Kok et al., supra.
Very little is known about the essential functions of Obg, however. To date, Obg has been validated to be essential for growth in both Gram-negative bacteria (E. coli, Caulobacter crescentus) and Gram-positive bacteria (Bacillus subtilis). Maddock et al., xe2x80x9cIdentification of an Essential Caulobacter crescentus Gene Encoding a Member of the Obg Family of GTP-Binding Proteinsxe2x80x9d, J. Bacteriol., Vol. 179, pp. 6426-31, 1997; Arigoni et al., xe2x80x9cA Genome-Based Approach for the Identification of Essential Bacterial Genesxe2x80x9d, Nature Biotechnology, Vol. 16, pp. 851-56, 1998; Trach et al., supra. In addition, depletion of Obg has been shown to cause cessation of Bacillus subtilis cell growth. Vidwans et al., xe2x80x9cPossible Role for the Essential GTP-Binding Protein Obg in Regulating the Initiation of Sporulation in Bacillus subtilisxe2x80x9d, J. Bacteriol., Vol. 177, pp. 3308-11, 1995. Because the Obg protein appears to be essential for cell growth and/or viability, compounds that interfere with Obg functionality, such as compounds which bind with and inhibit Obg, are of interest as potential antimicrobial agents.
The Obg family is attractive as a potential target for antibacterial drug discovery for several reasons. First, the obg gene is a novel target because it is a hypothetical open reading frame (ORF) and its function is essentially unknown. Further, Obg homologs are highly conserved among bacteria. Additionally, there is a low toxicity potential because the obg gene is distinguishable from its nearest human homologs. Furthermore, the obg gene encodes essential cell function, to the extent that mutation is detrimental for cell growth. Finally, Obg protein GTPase activity can be assayed in vitro, in light of functional similarity with in vivo activity, and assays are relatively simple for target development.
U.S. Pat. Nos. 5,585,277 and 5,679,582 to Bowie et al. disclose methods for screening chemical compounds for potential pharmaceutical or antimicrobial effectiveness. Among other things, these patents teach methods for identifying possible ligands which bind to target proteins. The methods of these patents may be useful in affinity-based assays for the initial identification of chemical compounds that interfere in vitro with protein function by binding with and inhibiting the protein of interest.
To date, however, the obg gene sequence and the encoded protein have not been identified in Staphylococcal bacterial strains, such as S. aureus. Accordingly, it can be seen that there remains a need in the art for the identification of GTPase obg gene DNA sequences that encode GTP-binding protein in Staphylococcal bacterial strains, such as S. aureus. Further, there remains a need in the art for the identification of a Staphylococcal obg gene that is capable of expression in a host cell to produce functional Staphylococcal GTP-binding protein for use in screening procedures for antimicrobial compounds. Additionally, there remains a need for recombinant expression vectors incorporating a Staphylococcal GTPase obg gene. Further, there remains a need for methods for producing GTP-binding protein and for using GTP-binding protein as a novel therapeutic target in screening procedures directed toward the discovery of antimicrobial agents active against Staphylococcal bacteria, and S. aureus in particular. The present invention is directed to meeting these needs.
It is an object of the present invention to provide a new and useful nucleotide sequence encoding Staphylococcal GTP-binding protein.
It is another object of the present invention to provide a Staphylococcal GTPase obg gene sequence.
It is a further object of the present invention to provide a novel S. aureus GTPase obg DNA sequence
It is yet another object of the present invention to provide a Staphylococcal GTPase Obg protein for use with antimicrobial compound screening methods.
A still further object is to provide a S. aureus GTP-binding protein amino acid sequence for use as a novel therapeutic target in affinity-based pharmacological screening procedures.
Yet another object of the present invention is to provide recombinant expression vectors incorporating the Staphylococcal GTPase obg gene of the present invention.
Still a further object of the present invention is to provide recombinant expression vectors that are useful in host cells, such as E. coli, for producing Staphylococcal GTP-binding protein.
It is still a further object of the present invention to provide methods for producing Staphylococcal GTP-binding protein that is functional in in vitro assays for identifying antimicrobial compounds active against Staphylococcal bacteria.
It is yet another object of the present invention to provide methods for using GTP-binding protein in affinity-based screening procedures for the identification of antimicrobial agents effective against Staphylococcal bacteria such as S. aureus. 
According to the present invention, an isolated polynucleotide that encodes a Staphylococcal GTP-binding protein is provided. More particularly, the polynucleotide encodes a Staphylococcus aureus GTP-binding protein. The isolated polynucleotide may particularly comprise a nucleotide sequence as set forth in SEQ ID NO:1, and may encode a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2. The isolated polynucleotide may comprise a Staphylococcal, and particularly a Staphylococcus aureus, GTPase obg gene. Variations of polynucleotides are contemplated, such as those comprising a complementary DNA strand, or which encode a fragment, derivative or analog of the polypeptide.
The present invention is also directed to an isolated and purified polypeptide comprising a Staphylococcal GTP-binding protein, and particularly a Staphylococcus aureus GTP-binding protein, such as a polypeptide encoded by an obg gene. In particular, the polypeptide may comprise an amino acid sequence as set forth in SEQ ID NO:2. The polypeptide may alternatively be a fragment, derivative or analog of a polypeptide.
The present invention additionally provides a recombinant expression vector comprising a polynucleotide that encodes a Staphylococcal GTP-binding protein, which may particularly be a Staphylococcus aureus GTP-binding protein. The expression vector may be a plasmid, and specifically a pET14b plasmid.
The present invention additionally pertains to an engineered host cell for use in producing Staphylococcal GTP-binding protein. The engineered host cell comprises an isolated polynucleotide that encodes a Staphylococcal GTP-binding protein, and particularly Staphylococcus aureus GTP-binding protein. The host cell may specifically be an E. coli bacterial cell, and the isolated polynucleotide may be introduced into the host cell by a vector, which may further include a regulatory sequence operatively linked to the isolated polynucleotide, such that expression of the isolated polynucleotide may be induced by addition of an inducing agent appropriate to the regulatory sequence.
The present invention further relates to a method of producing Staphylococcal GTP-binding protein, such as Staphylococcus aureus GTP-binding protein. The method broadly comprises the steps of introducing into suitable host cells a polynucleotide that encodes Staphylococcal GTP-binding protein, and culturing the host cells under conditions in which the host cells express the polynucleotide to produce Staphylococcal GTP-binding protein. The method may include the further step of recovering the Staphylococcal GTP-binding protein. The polynucleotide may be introduced into the host cells with a suitable expression vector, such as a plasmid. The host cells are preferably E. coli bacterial cells, such as E. coli BL21 (DE3).pLys.S cells. The method may include contacting the host cells with an inducing agent, such as isopropylthiogalactoside (IPTG), thereby to induce expression of the polynucleotide.
Finally, the present invention provides a method for high throughput screening to identify potential antimicrobial compounds useful against Staphylococcal bacterial strains. The steps of this method include providing a selected amount of Staphylococcal GTP-binding protein, contacting the Staphylococcal GTP-binding protein with a test compound to form a test combination, and determining whether the test compound binds with the Staphylococcal GTP-binding protein. A test compound that binds with the Staphylococcal GTP-binding protein is identified as a potential antimicrobial compound useful against Staphylococcal bacterial strains. The Staphylococcal GTP-binding protein may be a polypeptide having the sequence set forth in SEQ ID NO:2, or may be a fragment, derivative or analog thereof. The step of determining whether the test compound binds with the Staphylococcal GTP-binding protein may be accomplished by the steps of providing a control group of Staphylococcal GTP-binding protein, subjecting the test combination and control group to increasing temperature, and measuring the temperature at which biophysical catalyzation unfolding of the Staphylococcal GTP-binding protein occurs in each of the test combination and the control group. When unfolding of the Staphylococcal GTP-binding protein in the test combination occurs at a higher temperature than unfolding of the Staphylococcal GTP-binding protein in the control group, the test compound is, identified as a compound that binds with Staphylococcal GTP-binding protein.
These and other objects of the present invention will become more readily appreciated and understood from a consideration of the following detailed description of the exemplary embodiments of the present invention when taken together with the accompanying drawings, in which: